s2fieldsofstudy: Biology Chemistry

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Synthetic Mono-Carbonyl Curcumin Analogues Attenuate Oxidative Stress in Mouse Models

Alzheimer’s disease is the commonest form of dementia associated with short-term memory loss and impaired cognition and, worldwide, it is a growing health issue. A number of therapeutic strategies have been studied to design and develop an effective anti-Alzheimer drug. Curcumin has a wide spectrum of biological properties. In this regard, the antioxidant potentials of mono-carbonyl curcumin analogues (h1–h5) were investigated using in vitro antioxidant assays and hippocampal-based in vivo mouse models such as light–dark box, hole board, and Y-maze tests. In the in vitro assay, mono-carbonyl curcumin analogues h2 and h3 with methoxy and chloro-substituents, respectively, showed promising 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2, 2′-azinobis-3-ethylbenzothiazo-line-6-sulfonate (ABTS) free radical scavenging activities. In the in vivo studies, scopolamine administration significantly (p < 0.001) induced oxidative stress and memory impairment in mice, in comparison to the normal control group. The pretreatment with mono-carbonyl curcumin analogues, specifically h2 and h3, significantly decreased (123.71 ± 15.23 s (p < 0.001), n = 8; 156.53 ± 14.13 s (p < 0.001), n = 8) the duration of time spent in the light chamber and significantly enhanced (253.95 ± 19.05 s (p < 0.001), n = 8, and 239.57 ± 9.98 s (p < 0.001), n = 8) the time spent in the dark compartment in the light–dark box arena. The numbers of hole pokings were significantly (p < 0.001, n = 8) enhanced in the hole board test and substantially increased the percent spontaneous alternation performance (SAP %) in the Y-maze mouse models in comparison to the stress control group. In the biomarker analysis, the significant reduction in the lipid peroxidation (MDA) level and enhanced catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH) activities in the brain hippocampus reveal their antioxidant and memory enhancing potentials. However, further research is needed to find out the appropriate mechanism of reducing oxidative stress in pathological models.

Introduction
Alzheimer's disease (AD) is a neurological disorder linked with many clinical manifestations and a number of biomarkers and environmental and genetic factors are associated with the progression and development of this disease [1,2]. Alzheimer's disease (AD) is dants are common in various fruits, vegetables, and herbs [31], while synthetic antioxidants are butylated hydroxyl toluene (BHT), gallic acid ester, butylated hydroxyl anisole (BHA), and butylated hydroquinone [32]. Therefore, the design and development of safe and effective antioxidants are needed through quality research for the effective treatment of neurological disorders, including AD.
Curcumin, from the bright yellow dietary spice turmeric and found in the rhizome of Curcuma longa L. [33], possesses a variety of biological properties such as neuroprotective, antioxidant, hypoglycemic, ant-inflammatory, antimicrobial, antiviral, and anticancer, and is also widely used as a dietary pigment and spice [34][35][36][37][38]. Moreover, chronic administration of curcumin gives control over neurochemical and behavioral alterations reported in several preclinical and pharmacological animal models [39].
Based on these observations, our research group have previously reported on these compounds, indicating their neuroprotective potentials by inhibiting cholinesterases [5]. Keeping in mind their neuroprotective profile, this study was designed to find out their antioxidant potential in scopolamine-induced oxidative stress mouse models.

Chemicals and Animals
The chemical structures of the synthesized mono-carbonyl curcumin analogues (h1-h5) used in this study are illustrated in Figure 1, as previously reported by our research group [5]. All the chemicals and reagents used in this research, including DPPH, ABTS, methanol, acetic acid, n-butanol, trichloracetic acid (TCA), reduced nicotinamide adenine dinucleotide (NADH), thiobarbituric acid (TBA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), were of analytical grade from Sigma-Aldrich (Merck, Darmstadt, Germany) and purchased from the local market. Scopolamine (butylbromide) and donepezil were obtained from Venous Pharma Pvt. Lahore and Platinum Pharmaceuticals Pvt. Karachi, Pakistan, respectively.
Three-month-old mice (Balb/C) weighing 19-23 gm of both sexes were procured from the "National Institute of Health (NIH), Islamabad". Mice were housed under a 12 h light and 12 h dark cycle with free access to food and water at 25 ± 3 °C and 55-65% relative humidity in the animal house. All the animals were acclimatized to the laboratory environment for two weeks before running the experimental models. This study was performed as per the approval of the "Departmental Ethical Committee" vide notification (SBBU/IEC-20-02) in accordance with the "Scientific Procedure Issue-I" 2008 animal bylaws of the University of Malakand. All the chemicals and reagents used in this research, including DPPH, ABTS, methanol, acetic acid, n-butanol, trichloracetic acid (TCA), reduced nicotinamide adenine dinucleotide (NADH), thiobarbituric acid (TBA), catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), were of analytical grade from Sigma-Aldrich (Merck, Darmstadt, Germany) and purchased from the local market. Scopolamine (butylbromide) and donepezil were obtained from Venous Pharma Pvt. Lahore and Platinum Pharmaceuticals Pvt. Karachi, Pakistan, respectively.

In Vitro Antioxidant Activity
Three-month-old mice (Balb/C) weighing 19-23 gm of both sexes were procured from the "National Institute of Health (NIH), Islamabad". Mice were housed under a 12 h light and 12 h dark cycle with free access to food and water at 25 ± 3 • C and 55-65% relative humidity in the animal house. All the animals were acclimatized to the laboratory environment for two weeks before running the experimental models. This study was performed as per the approval of the "Departmental Ethical Committee" vide notification (SBBU/IEC-20-02) in accordance with the "Scientific Procedure Issue-I" 2008 animal bylaws of the University of Malakand.

In Vitro Antioxidant Activity
In vitro antioxidant profiles of mono-carbonyl curcumin analogues (h1-h5) were explored according to the Brand-Williams et al. protocol of 1995 [40], by means of DPPH and ABTS as free radicals. In the assay of DPPH, the tested compounds (h1-h5) and the standard drug tocopherol were mixed with 31.25-1000 µg/mL concentrations of DPPH solution, and a spectrophotometer at 517 nm was run and the absorbance was recorded. Similarly, the ABTS assay was performed for the tested compounds (h1-h5) and standard drug in a similar concentration. In this, 0.1 mL of sample and standard solution were mixed with ABTS at the same concentrations and absorbance at 734 nm was noted. Then, IC 50 values for the tested samples were calculated.

Acute Toxicity Study
The safety of these compounds was ensured by conducting acute toxicity studies in mice to find any possible toxicological properties and select the safe optimum dose of the tested compounds (h1-h5) for conducting the in vivo studies. The mono-carbonyl curcumin analogues (h1-h5) were orally administered to different groups of mice in doses of 5,15,30,50,75, 100, and 150 mg/kg body weight, and all the animals were keenly observed for toxicity indications for 24 h such as convulsions, tremors, motor activity, loss of righting reflex, lacrimation, diarrhea, muscle spasm, salivation, sedation, and hypnosis. Then, animals were further observed for mortality for 72 h. The animals were safe up to a 150 mg/kg body weight dose. Therefore, as per OECD guidelines, a 15 mg/kg dose of the tested samples as 1/10th of 150 mg/kg was chosen as a suitable dose for in vivo studies of the mono-carbonyl curcumin analogues (h1-h5) [6].

Experimental Design and Animal Dosing
All the animals were divided into eight groups, having eight in each (n = 8) and treated as the sample-treated groups, stress control group, standard treatment group, and normal control group for seven days. In this study, the normal control group was given 5 mL/kg (p.o) normal saline; the stress control group was administered 5 mL/kg (p.o) normal saline (2% Tween 80); the standard control group received donepezil 2 mg/kg (p.o); and the sample-treated groups received mono-carbonyl curcumin analogues (h1-h5) at 15 mg/kg body weight (p.o) suspended in 2% Tween 80, for seven days. On day seven, 30 min after the last dose, scopolamine 1 mg/kg (i.p.) was administered to all groups except the normal control group. Then, the in vivo behavioral activities were performed accordingly.

Behavioral Studies
Oxidative stress was induced in mice using scopolamine in the behavioral mouse models, including light-dark box, hole board, and Y-maze mouse models.

Light-Dark Box
The paradigm of the light-dark box was used for the investigation of behavioral and learning tasks using the mono-carbonyl curcumin analogues (h1-h5) according to the standard procedure [41]. The test was based on the principle that mice favor the dark compartment. This paradigm consists of two compartments and is made of polyacrylic sheets, with a larger transparent chamber with dimensions of 30 cm × 30 cm × 35 cm attached in parallel position with a smaller dark chamber colored with black and having dimensions of 20 cm × 30 cm × 35 cm. A small opening of 5 cm × 5 cm was made for entrance in the middle of the separating wall of both chambers. Lines, 1 mm apart, were drawn on the floor of both chambers. The mouse was observed for 5 min after being put in the light compartment and the time spent in each chamber was noted. The doses were administered to each mouse accordingly, and after the completion of the last dose, the time spent in each compartment was recorded for two days [42].

Hole Board Test
The hole board paradigm was used for the investigation of the learning behavior with the mono-carbonyl curcumin analogues (h1-h5) according to the standard procedure [43]. The paradigm of the hole board was designed with a rectangular shape from polyacrylic sheets with dimensions of 35 cm × 45 cm × 45 cm. There were sixteen holes (2 cm in diameter) containing black colored sheets 5 cm above the bottom, which were connected to the internal corners of the box. The numbers of hole pokings were recorded for 5 min after putting the mouse in the middle of the apparatus. After completion of the last dose, animals were observed for two consecutive days [44].

Y-Maze Test
The mono-carbonyl curcumin analogues (h1-h5) were investigated for attenuation of memory impairment with the help of the Y-maze behavioral mouse model according to the standard protocol [5]. In this test, oxidative stress was induced by injecting 1 mg/kg (i.p.) scopolamine. The paradigm of the Y-maze mice model consists of three equal arms linked with each other at an angle of 120 • and designed in a Y-shape, label as A, B, and C for convenience. The arms were 20 cm long, 6 cm wide, and 15.5 cm high. Mice were placed at the center of one arm and allowed to freely explore the apparatus for a period of 5 min. The number of arm entries executed by each mouse was recorded. A complete arm entry is achieved when the hind paws of the animal are totally inside in one of the arms while alternation involved entries made consecutively around the three arms. A solution of ethanol 70% was applied to the Y-maze arena to make it clean and to avoid olfactory cues after every run. The escape latency (seconds) at the end of day seven was noted for each animal. The spontaneous alternation performance (SAP%) was calculated by recording same-arm returns (SARs), alternate arm returns (AARs), and the number of arm entries [6].

Assessment of Biochemical Parameters and Biomarker Study
The biomarker approach was used to find out the antioxidant potential of the monocarbonyl curcumin analogues (h1-h5) in oxidative stress, induced by scopolamine with alteration in the endogenous antioxidant enzyme system. Soon after the behavioral analysis, mice were sacrificed by cervical dislocation for a painless death, and the brain tissues were isolated and hippocampus was obtained after homogenization. The hippocampus part of brain tissue was chilled before biomarker analysis in phosphate buffer solution [45].

Measurement of Malondialdehyde (MDA) Level
The malondialdehyde level in the hippocampus was investigated according to the standard procedure [46]. In this method, 100 µL tissue homogenate was added to the mixture of 1.5 mL of thiobarbituric acid (TBA) 0.8%, 1.5 mL of (20%) acetic acid, and 200 µL (8%) sodium dodecyl sulfates. It was then heated for an hour at 90 • C, cooled to room temperature, and added to 5 mL of n-butanol. The mixture was centrifuged for 10 min at 976× g and the organic layer was separated out. The MDA level was recorded by measuring absorbance at 532 nm [44].

Catalase (CAT) Activity
The catalase activity was measured in the hippocampus according to the reported procedure of Sinha in 1972 with little modification [47]. Tissue homogenate 0.1 mL was mixed with 1 mL (0.01 M) phosphate buffer pH 7, 5% potassium dichromate-acetic acid 1:3, H 2 O 2 (2 M), and 2 mL of dichromate acetic acid. The absorbance was measured at 620 nm and the catalase activity was expressed in the µM of H 2 O 2 decomposing/min/mg of tissue protein [44].

Superoxide Dismutase (SOD) Activity
The superoxide dismutase activity of the mono-carbonyl curcumin analogues was measured according to the standard procedure by Kakkar et al., in 1984 [48], in the mouse hippocampus. The brain tissue homogenate (0.5 mL) dilution was made in 1 mL of distilled water and then shaken with 2.5 mL of ethanol and 1.5 mL of chloroform. The mixture was centrifuged at 4 • C for a period of 1 min. The supernatant was mixed with 1.2 mL (0.025 M) sodium pyrophosphate buffer of 8.4 pH, 0.3 mL (30 µM) NBT, 3 mL distilled water, 0.1 mL (186 µM) PMS, and 0.2 mL (780 µM) NADH. It was then incubated at 30 • C for 90 s and, finally, 1 mL acetic acid was added to stop the reaction. The mixture was stirred vigorously, n-butanol was added, and it was stirred again. Then, the butanol layer was separated out. The absorbance of butanol was measured at 560 nm. The measured SOD amount was presented as a unit/mg of protein [44]. The effect of mono-curcumin analogues (h1-h5) on glutathione activity was investigated in the mouse hippocampus according to the reported procedure by Moron et al. in 1979 [49]. First, 0.4 mL (20%) TCA was added to 0.4 mL of the homogenate and centrifuged for 20 min at 10,000× g at 4 • C. The supernatant (0.25 mL) was added to 0.6 M (2 mL) DTNB and phosphate buffer pH 8.0 (0.2 M), and the final volume was 3 mL. The GSH activity was recorded at 412 nm absorbance and expressed as µM/mg of protein [44].

Statistical Analysis
Results are presented as mean ± SD, a Kolmogorov-Smirnov test was applied for normality distribution of each data set and one-way ANOVA was used followed by Bonferroni's multiple comparison tests (post hoc) using statistical package SPSS, version 25. The value of p = 0.05 was designated as significant.

In Vitro Antioxidant Activity
Results of the in vitro antioxidant assay of mono-carbonyl curcumin analogues (h1-h5) are shown in Table 1. In this assay, the mono-carbonyl curcumin analogues h2 and h3 with IC 50 values of 53.29 ± 2.13 and 82.43 ± 2.17 µg/mL, respectively, showed maximum response against DPPH free radicals in comparison to the positive control with an IC 50 value of 9.16 ± 1.14 µg/mL. Among other curcumin analogues, h1 showed moderate free radical scavenging activity with IC 50 160.32 ± 1.37 µg/mL, while, h4 and h5 showed weaker antioxidant response against DPPH free radicals. Similarly, in the ABTS assay, h2 with an IC 50 value of 70.21 ± 1.26 µg/mL showed significant antioxidant response in comparison to the standard drug tocopherol, having an IC 50 of 13.18 ± 1.16 µg/mL, and h3 exhibited IC 50 of 143.65 ± 2.43 µg/mL, showing weaker antioxidant activity, while h1, h4, and h5 showed a poor response. Data are presented as mean ± SEM (n = 3), the significantly different values were compared to the positive control.

Acute Toxicity
The mono-carbonyl curcumin analogues (h1-h5) were investigated for possible toxicological effects, and it was observed that up to a maximum administered dose of 150 mg/kg body weight, there was no toxicological response or mortality in mice. After acute toxicity screening, 15 mg/kg b.w. was selected as a suitable dose for the in vivo studies.

Light-Dark Box Apparatus
The effects of mono-carbonyl curcumin analogues (h1-h5) on the mice's memory were investigated by using a light-dark box apparatus and the results are shown in Figure 2. Mice in the normal control group stayed for less time in the light chamber and stayed for a longer period in the dark compartment on day 1. Animals in the stress control (amnesic) group remained for a significantly (p < 0.001) longer time in the light compartment and spent less time in the dark compartment in comparison to the normal control group, indicating induction of stress. Mice in the treatment groups, treated specifically with the standard drug, and compounds h2, h3, and h4, spent significantly (p < 0.001) more time in the dark compartment in comparison to the stress control group, which showed memory improvement by reducing stress (Figure 2A,B). Those treated with compound h1 spent comparatively (p < 0.01) more time in the dark arena and showed memory enhancement effects as compared to the stress control group, while h5 showed a poor (p < 0.05) response on day 1. Similarly, on day 2, those treated with compounds h2 (p < 0.001), h3 (p < 0.001), h1 (p < 0.01), h4, and h5 (p >0.05) remained for a short period in the light chamber and those treated with compounds h2 (p < 0.001) and h3 (p < 0.01) spent significantly more time in the dark chamber, revealing a memory enhancement effect as compared to the stress control group, while h1, h4, h5 (p > 0.05) showed a poor response ( Figure 2C,D).
The effects of mono-carbonyl curcumin analogues (h1-h5) on the mice's memory were investigated by using a light-dark box apparatus and the results are shown in Figure 2. Mice in the normal control group stayed for less time in the light chamber and stayed for a longer period in the dark compartment on day 1. Animals in the stress control (amnesic) group remained for a significantly (p < 0.001) longer time in the light compartment and spent less time in the dark compartment in comparison to the normal control group, indicating induction of stress. Mice in the treatment groups, treated specifically with the standard drug, and compounds h2, h3, and h4, spent significantly (p < 0.001) more time in the dark compartment in comparison to the stress control group, which showed memory improvement by reducing stress (Figure 2A,B). Those treated with compound h1 spent comparatively (p < 0.01) more time in the dark arena and showed memory enhancement effects as compared to the stress control group, while h5 showed a poor (p < 0.05) response on day 1. Similarly, on day 2, those treated with compounds h2 (p < 0.001), h3 (p < 0.001), h1 (p < 0.01), h4, and h5 (p ˃ 0.05) remained for a short period in the light chamber and those treated with compounds h2 (p < 0.001) and h3 (p < 0.01) spent significantly more time in the dark chamber, revealing a memory enhancement effect as compared to the stress control group, while h1, h4, h5 (p ˃ 0.05) showed a poor response ( Figure 2C,D). shown as mean ± SD, n = 8. Kolmogorov-Smirnov test was applied for normality distribution and each data set was found to have a normal distribution. One-way ANOVA, followed by Bonferroni's multiple comparison tests (post hoc), was applied and expressed as ### p < 0.001 vs. normal control group, and *** p < 0.001, ** p < 0.01, * p < 0.05, "ns" p ˃ 0.05 vs. stress control group.

Hole Board Assay
The numbers of hole pokings were significantly (p < 0.001) decreased by the administration of scopolamine in comparison to the normal control group, which showed oxidative stress (amnesia) in the stress control group on days 1 and 2, as shown in Figure  3A,B. The mice in the standard control group showed a significantly increased number of shown as mean ± SD, n = 8. Kolmogorov-Smirnov test was applied for normality distribution and each data set was found to have a normal distribution. One-way ANOVA, followed by Bonferroni's multiple comparison tests (post hoc), was applied and expressed as ### p < 0.001 vs. normal control group, and *** p < 0.001, ** p < 0.01, * p < 0.05, "ns" p >0.05 vs. stress control group.

Hole Board Assay
The numbers of hole pokings were significantly (p < 0.001) decreased by the administration of scopolamine in comparison to the normal control group, which showed oxidative stress (amnesia) in the stress control group on days 1 and 2, as shown in Figure 3A,B. The mice in the standard control group showed a significantly increased number of hole pokings (p < 0.001) on day 1 and (p < 0.01) on day 2, showing a reduction in amnesia. Similarly, compound h2 significantly (p < 0.01) increased the number of hole pokings, followed by h1, h3, h4 (p < 0.05), indicating its antiamnesic potentials, while h5 (p > 0.05) showed no prominent response on day 1. On day 2, only compound h2 (p < 0.05) comparatively increased the number of hole pokings while the rest of the compounds showed no prominent effects on hole pokings in comparison to the stress control group. Results are shown as mean ± SD, n = 8. Kolmogorov-Smirnov test was applied for normality distribution and each data set was found to have a normal distribution. One-way ANOVA, followed by Bonferroni's multiple comparison tests (post hoc), was applied on each data set and expressed as ### p < 0.001 vs. normal control group, and *** p < 0.001, ** p < 0.01, * p < 0.05, "ns" p ˃ 0.05 vs. stress control group.

Y-Maze Test
The mono-carbonyl curcumin analogues (h1-h5) were investigated for memory-enhancing effects induced by scopolamine in the Y-maze mouse model ( Figure  4). In the stress control group, scopolamine 1 mg/kg (i.p.) administration caused significant (p < 0.001) memory deficits in the stress control group. The standard control and compounds h2 (p < 0.001) and h3 (p < 0.001) significantly reversed the scopolamine-induced memory deficits by enhancing the percent spontaneous alternation performance in mice. Similarly, compounds h1, h4, and h5 (p ˃ 0.05) showed poor response in comparison to the stress control group. The results indicated that mono-carbonyl curcumin analogues, specifically h2 and h3, showed memory-enhancing effects by reversing the memory deficits caused by scopolamine administration. Results are shown as mean ± SD, n = 8. Kolmogorov-Smirnov test was applied for normality distribution and each data set was found to have a normal distribution. One-way ANOVA, followed by Bonferroni's multiple comparison tests (post hoc), was applied on each data set and expressed as ### p < 0.001 vs. normal control group, and *** p < 0.001, ** p < 0.01, * p < 0.05, "ns" p >0.05 vs. stress control group.

Y-Maze Test
The mono-carbonyl curcumin analogues (h1-h5) were investigated for memoryenhancing effects induced by scopolamine in the Y-maze mouse model (Figure 4). In the stress control group, scopolamine 1 mg/kg (i.p.) administration caused significant (p < 0.001) memory deficits in the stress control group. The standard control and compounds h2 (p < 0.001) and h3 (p < 0.001) significantly reversed the scopolamine-induced memory deficits by enhancing the percent spontaneous alternation performance in mice. Similarly, compounds h1, h4, and h5 (p > 0.05) showed poor response in comparison to the stress control group. The results indicated that mono-carbonyl curcumin analogues, specifically h2 and h3, showed memory-enhancing effects by reversing the memory deficits caused by scopolamine administration. compounds h2 (p < 0.001) and h3 (p < 0.001) significantly reversed the scopolamine-induced memory deficits by enhancing the percent spontaneous alternation performance in mice. Similarly, compounds h1, h4, and h5 (p ˃ 0.05) showed poor response in comparison to the stress control group. The results indicated that mono-carbonyl curcumin analogues, specifically h2 and h3, showed memory-enhancing effects by reversing the memory deficits caused by scopolamine administration. Kolmogorov-Smirnov test was applied for normality distribution and each data set was found to have a normal distribution. One-way ANOVA, followed by Bonferroni's multiple comparison tests (post hoc), was applied on each data set and expressed as ### p < 0.001 vs. normal control group, and *** p < 0.001, "ns" p >0.05 vs. stress control group.

Assessment of Biochemical Parameters and Biomarker Study
The mono-carbonyl curcumin analogues (h1-h5) after the in vivo behavioral studies were immediately subjected to biochemical assessment and biomarker studies. The isolated, homogenized hippocampus was analyzed for the determination of the activity of antioxidant enzymes and the level of malondialdehyde. In this study, scopolamine administration caused a reduction in the antioxidant enzymes and increased the MDA level, which indicated the induction of oxidative stress ( Figure 5).
The administration of scopolamine 1 mg/kg (i.p.) induced oxidative stress and caused a significant (p < 0.001) increase in the lipid peroxidation (MDA) level in comparison to the normal control group ( Figure 5A). Pretreatment with mono-carbonyl curcumin analogues for seven days reversed the oxidative stress by reducing the MDA level. The standard drug donepezil and h2 significantly (p < 0.001) decreased the MDA level in comparison to the stress control group. Curcumin analogue h3 moderately (p < 0.05) reduced the MDA level as compared to the stress control group. Likewise, in this assay, compounds h1, h4, and h5 showed no promising response.
In the catalase (CAT) assay, injecting scopolamine 1 mg/kg (i.p.) induced oxidative stress in the mice of the stress control group by significantly (p < 0.001) decreasing the activity of catalase in the hippocampus in comparison to the normal control group ( Figure 5B). Pretreatment with compounds h1, h2, h3 and standard control significantly (p < 0.001) increased the catalase activity in the hippocampus in comparison to the stress control group, indicating their antioxidant potential. Compounds h4 and h5 did not show a significant (p > 0.05) increase in catalase level and showed comparative antioxidant response as compared to the stress control group, while h4 and h5 showed no promising (p > 0.05) antioxidant activity. Results are shown as mean ± SD, n = 8. Kolmogorov-Smirnov test was applied for normality distribution and each data set was found to have a normal distribution. One-way ANOVA, followed by Bonferroni's multiple comparison tests (post hoc), was applied on each data set and expressed as ### p < 0.001 vs. normal control group, and *** p < 0.001, ** p < 0.01, * p < 0.05, "ns" p ˃ 0.05 vs. stress control group.
Similarly, in the assay of glutathione (GSH), the activity of GSH was significantly (p < 0.001) lower upon the administration of scopolamine in the hippocampus of mice in the stress control group in comparison to the normal control group, indicating induction of oxidative stress ( Figure 5D). The standard (p < 0.001) and the mono-carbonyl curcumin analogues h2 (p < 0.001) and h3 (p < 0.001) significantly increased the GSH activity in the hippocampus in comparison to the stress control group, which indicated the reversal of oxidative stress and showed the antioxidant potential of these compounds in mouse models, while compounds h1, h4, and h5 (p ˃ 0.05) showed no promising antioxidant responses.

Discussion
The reactive oxygen species caused oxidative stress which is responsible for neurodegenerative diseases [50], and it has been reported that oxidative stress plays a pivotal Results are shown as mean ± SD, n = 8. Kolmogorov-Smirnov test was applied for normality distribution and each data set was found to have a normal distribution. One-way ANOVA, followed by Bonferroni's multiple comparison tests (post hoc), was applied on each data set and expressed as ### p < 0.001 vs. normal control group, and *** p < 0.001, ** p < 0.01, * p < 0.05, "ns" p >0.05 vs. stress control group.
The study of superoxide dismutase (SOD) in the hippocampus of the mouse brain indicated that upon administration of scopolamine 1 mg/kg (i.p.), oxidative stress was induced in the hippocampus in the animals of the stress control group ( Figure 5C). This revealed the significant (p < 0.001) reduction in SOD activity, which reverted the memory deficits with the pretreatment of consecutive doses for seven days of the standard drug (p < 0.001), h2 (p < 0.001), and h3 (p < 0.01), in comparison to the stress control group, indicating that these types of curcumin analogues with methoxy and chloro substituent have strong antioxidant potential, while compound h1 with methyl, h4 with N,N dimethyl substituent, and h5 with nitro substituent showed no promising (p > 0.05) antioxidant activity in comparison to the stress control group.
Similarly, in the assay of glutathione (GSH), the activity of GSH was significantly (p < 0.001) lower upon the administration of scopolamine in the hippocampus of mice in the stress control group in comparison to the normal control group, indicating induction of oxidative stress ( Figure 5D). The standard (p < 0.001) and the mono-carbonyl curcumin analogues h2 (p < 0.001) and h3 (p < 0.001) significantly increased the GSH activity in the hippocampus in comparison to the stress control group, which indicated the reversal of oxidative stress and showed the antioxidant potential of these compounds in mouse models, while compounds h1, h4, and h5 (p > 0.05) showed no promising antioxidant responses.

Discussion
The reactive oxygen species caused oxidative stress which is responsible for neurodegenerative diseases [50], and it has been reported that oxidative stress plays a pivotal role as one of the etiologic factors of dementia and more than a hundred other diseases [44,51]. The main causes of memory loss in Alzheimer's disease (AD) are severe oxidative damage and alterations in the cholinergic system in the brain, deposition of β-amyloid protein, and extra neuronal plaque development [44]. Scopolamine, a muscarinic ACh receptor blocker, is extensively used in rodents and humans to induce oxidative stress, alter the neuronal cholinergic pathway, and reduce hippocampal volume [22]. Scopolamine quickly crosses the blood-brain barrier, causing induction of antimuscarinic activity by depleting acetylcholine, leading to memory loss. Thus, scopolamine is widely used for the induction of dementia in experimental animal models to investigate new compounds for assessing memory and learning behaviors [24].
Those agents which prevent oxidative stress and increase cholinergic activity in the neurons of the brain could be used to reduce the severity of dementia [52]. The symptomatic relief in dementia is provided by activating the cholinergic system by using cholinesterase blockers. At present, galantamine, donepezil, tacrine, and rivastigmine are the approved cholinesterase inhibitors recently reported to provide symptomatic relief in AD [6,28,29]. These only manage the severity of the illness and do not cure the disease completely due to their diminished long-term efficacy and are often associated with side effects. Therefore, the design and development of an effective agent for the treatment of AD are urgently required [6]. In this scenario, our initial study reported on mono-carbonyl curcumin analogues (h1-h5) proven to be effective acetylcholine esterase inhibitors in animal models [5]. These compounds showed significant antioxidant potential in the in vitro assay. Research studies indicated that methoxy (-OCH 3 ) and hydroxyl (-OH) substituted derivatives are responsible for the their antioxidant effects [53]. The monocarbonyl curcumin analogues with a methoxy substituted moiety exhibited significant antioxidant potential and this was consistent with reported studies [54,55].
The light-dark box mouse model works on the principle of the animal behavior of avoiding the light chamber and exploring the novel environment [56]. The memory assessment using the Y-maze test works on the principle of rodents exploring objects and is considered one of the most convenient and reliable behavior models [57]. The Y-maze mouse model records the percent of spontaneous alternation behavior in mice [6]. Similarly, the head dips and rearing (hole poking) behaviors in mice in the hole board test were used for the assessment of anxiety-like behavior [58,59]. The time spent in the light chamber was longer upon administration of scopolamine, indicating the induction of oxidative stress. The reduced tendency of animals to enter the light compartment, the decrease in the number of hole pokings, and the reduction in the percent spontaneous alternation performance represent a condition of fear, anxiety, and stress in the light-dark box, hole board, and Y-maze mouse models, respectively [5,41,60]. The mono-carbonyl curcumin analogues in the in vivo models significantly decreased the time spent in the light chamber and increased the time spent in the dark compartment in the light-dark box test. The numbers of holes pokings were higher in the hole board test and the percent spontaneous alternation performance was higher in the Y-maze test. The reported curcumin analogues showed promising results and increased the percent spontaneous alternation performance. These results were consistent with previous findings [6,61].
The administration of scopolamine was responsible for the brain's reduced antioxidant capacity and a marked increase in the MDA level, which is considered the index of lipid peroxidation. Scopolamine caused an increase in the markers of oxidative stress and memory impairment by reducing the cholinergic neurotransmission in the brain [62]. It has been reported that during stressful situations the nervous system is often prone to enhanced lipid peroxidation levels due to high oxygen tension and oxidative damage [45]. In addition, injecting scopolamine in rodents weakens the antioxidant system and induces the lipid peroxidation in the brain, including in those brain parts responsible for memory and learning phenomena [63]. In conditions of severe oxidative stress, the increased generation of reactive oxygen species interacts with many vital components of cells, including lipids, carbohydrates, nucleic acids, and proteins, causing damage to neuronal cells, leading to memory impairment [24]. Similarly, our research showed that the standard drug, donepezil, significantly attenuated the oxidative stress by protecting the antioxidant system and this was consistent with reported studies [64]. These outcomes also suggest the significant antioxidant potential of donepezil, which is important for the attenuation of oxidative stress related to neurodegenerative disorders [65].
In this study, oxidative stress was induced by injecting scopolamine, appearing in the form of disorderly markers of oxidative stress such as higher lipid peroxidation (MDA) and a reduction in the antioxidant enzyme system catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH), responsible for memory loss [62]. The more oxygen is consumed, the more the nervous system is prone to enhanced MDA levels during stressful circumstances [45,66]. The reduction in lipid peroxidation with pretreatment of monocarbonyl curcumin analogues indicated their antioxidant potential by reducing oxidative stress in mice. It has been reported in various studies that administration of curcumin causes a reduction in oxidative stress [38,67]. The scopolamine administration not only increased the lipid peroxidation but also reduced the activity of antioxidant systems in the brain such as CAT, SOD, and GSH [22,68]. The reported mono-carbonyl curcumin analogues substantially altered the effect of scopolamine and increased the activity of CAT, SOD, and GSH by reducing the oxidative stress in the hippocampus, and these findings were consistent with a reported study [38]. At present, treating Alzheimer's disease and other neurological disorders with curcumin is a focus of interest for researchers [37]. Chronic administration of curcumin analogues improves neurogenesis [69]. Bearing in mind the concluding results of this work, mono-carbonyl curcumin analogues reduce the severity of dementia as well as improve cognition.
Despite its tremendous pharmacological profile, curcumin has poor water solubility, low permeability through the BBB, and poor bioavailability, which will make it difficult for it to be an ideal drug [70]. Our reported curcumin analogues have also been found to be poorly water soluble. The low bioavailability of curcumin is considered one of the main obstacles for its clinical application against various disorders such as AD and cancers [71]. There are various strategies, such as nano-formulations [72] and the synthesis of curcumin analogues with structural modification and also combination with other multiple components, to enhance its efficacy. These strategies will not only improve the efficacy of curcumin and its analogues but will also enhance its therapeutic effectiveness against a number of diseases such as AD.

Limitations
The current research work involved in vitro and hippocampal-based investigation. Further research studies are suggested to explore the complete pharmacological profiles, specifically neuropharmacological, of these compounds to find a molecule that can be added to the therapy of oxidative stress-induced neurodegenerative diseases such as Alzheimer's disease (AD).

Conclusions
In conclusion, oxidative stress is responsible for altering the endogenous antioxidant markers and is associated with reduced cholinergic system activity, leading to dementia. The mono-carbonyl curcumin analogues decrease oxidative stress and show their effective-ness in attenuating memory loss associated with dementia in mice. This study suggested that the neuroprotective effect of these compounds on scopolamine-induced oxidative stress was due to improving the antioxidant and cholinergic systems in the hippocampus. In addition, these compounds might be appropriate candidates for treating stress-induced cognitive dysfunction after exploring their complete pharmacological profile.


Ankle joint flexibility affects undulatory underwater swimming speed

The movement of undulatory underwater swimming (UUS), a swimming technique adapted from whales, is mainly limited by human anatomy. A greater ankle joint flexibility could improve the imitation of the whale's flap of the fin and therefore enhance USS performance. The aim of this study was to investigate the impact of ankle joint flexibility on swimming velocity and kick efficiency during UUS by comparing kinematics of swimming trials with reduced, normal, and enhanced maximum angles of plantar flexion. Ten well trained swimmers (5m and 5f; 22 ± 4years; 177 ± 7cm; 74 ± 15kg), performed multiple trials of UUS with normal, restricted, and increased ankle joint flexibility on two separate days in randomized order. Kick frequency was controlled by a metronome. Plantar flexion (PF) was restricted by tape application on both feet and increased by passive-dynamic stretching. All trials were filmed. Kinematics were obtained with two-dimensional motion analysis. Tape application restricted maximum PF by 10.42% while stretching increased PF by 6.87% compared to normal PF. Swimming velocity and kick efficiency significantly decreased during swimming with restricted PF (1.13 ± 0.13m*s−1; 0.69 ± 0.09m) compared to normal (1.20 ± 0.14 m*s−1; 0.72 ± 0.10m) and increased (1.22 ± 0.15m*s−1; 0.73 ± 0.10m) PF. Swimming velocity and kick efficiency did not differ between normal and increased PF. Body height normalized swimming velocity correlated significantly with PF angle (r = 0.538). The results suggest that UUS velocity is affected by impaired PF. Particularly swimmers with low or average maximum PF angles may benefit from a long-term ankle joint flexibility program to improve their UUS performance.

Introduction
In competitive swimming races, success and failure are often discriminated by milliseconds only (McCullough et al., 2009;Yuan et al., 2019). As a result, biomechanical characteristics affecting swimming stroke efficiency must be identified and optimized to improve the athlete's performances even by marginal gains. Since undulatory underwater swimming (UUS) is often faster than the main swimming strokes (Bissig et al., 2004;Ungerechts et al., 2009;Schneider, 2012), improving dolphin kick efficiency has the . /fspor. . potential to improve overall competitive swimming times (Nakashima, 2009;Gonjo and Olstad, 2020). The more streamlined body posture (Schneider, 2012;Zamparo et al., 2012), as well as the smaller up to non-existing wave resistance underneath the water surface allow to maintain the gliding speed after start and turns as long as possible and to reduce the deceleration during the diving phase (Zamparo et al., 2012). Although the optimal distance traveled underwater seems to be individually different and depends on the following swimming stroke (Veiga and Roig, 2015;Morais et al., 2019), studies have shown that faster swimmers had longer UUS distances (Veiga et al., 2014) and UUS can have a positive impact on swimming velocity and stroking length on start and turn segments (Veiga and Roig, 2016) as well as total race times (Morais et al., 2019). Thus, the permitted diving distance of 15 m (Fédération internationale de Natation, 2017), which equates to 30% of a long course and 60% of a short course, provides an opportunity to improve overall competitive swimming times by enhancing UUS performance.
To maximize the speed of UUS, the optimized movement must be adapted to human anatomy (Hochstein and Blickhan, 2014). Since the human body has only a few joints that can execute the undulatory movement (hip, knee and ankle), the smooth transition of the body wave is highly limited and the propulsion effect is quite low compared to whales with a larger number of separate joints (Von Loebbecke et al., 2009a,b). However, a greater ankle joint flexibility which allows increased plantar flexion may improve the dolphin kick performance as the greatest propulsion is generated with the kicking movement of the feet (Von Loebbecke et al., 2009b). More flexible ankle joints could thus superiorly imitate the efficient kicking movement of a fin (Reischle, 1988;Wick, 2013). The displacement of water during the down kick would be directed rather backwards than downwards, so there would be a higher propulsion with the same power efficiency (McCullough et al., 2009;Hochstein et al., 2010;Schneider, 2012;Séhel, 2016). Furthermore, the greater range of motion could increase the flipping movement of the feet which would enhance the usable propulsive momentum by faster reversion of the vortices (Strass et al., 2002;Ungerechts et al., 2009). Additionally, a greater ankle joint flexibility could result in a more harmonized and energy-efficient undulatory movement (Hahn, 2013). This could enhance the kicking frequency which, in turn, is positively correlated to swimming speed (Arellano et al., 2003;McCullough et al., 2009). Therefore, elite as well as recreational athletes could improve their swimming performance in different strokes via more efficient UUS by enhancing their ankle joint flexibility.
The potential benefit of more flexible ankle joints on kicking efficiency and swimming velocity is often mentioned but rarely directly tested. Only a few studies investigated the effect of the ankle joint flexibility on swimming velocity during UUS (Sugimoto et al., 2008;Willems et al., 2014;Connaboy et al., 2016;Shimojo et al., 2019;Wadrzyk et al., 2019). Different methodological approaches (e. g., different swimming distances, different number of analyzed swimming trials and swimming cycles per trial, different magnitude of ankle joint flexibility restriction, dimension of filming and analysis) complicate the direct comparison of the results. A restriction of the plantar flexion (PF) angle by tape application consistently decreased swimming velocity, however, it remained unclear if an increase in the range of ankle movement would enhance the swimming velocity.
The aim of this study was to investigate the impact of ankle joint flexibility on swimming velocity and kick efficiency during UUS by comparing kinematics of swimming trials with different maximum angles of plantar flexion. We hypothesized that a greater ankle joint flexibility (maximum PF angle) is associated with a greater swimming velocity and kick efficiency.

Subjects
Five male and five female swimmers (age: 22.00 ± 4.19 years, height: 176.90 ± 6.64 cm, weight: 74.20 ± 15.11 kg, training experience: 15.7 ± 4.0 years) were tested within this study. Swimmers who reported former ankle surgery or structural ankle injuries were excluded from participation in this study. The study was reviewed and approved by the Ethics Committee of the Faculty of Humanities and Social Sciences of the Humboldt-Universität zu Berlin. The participants provided their written informed consent to participate in this study.

Experimental setup
Each participant got tested on two separate days at an interval of a week. Anthropometric data (body height and weight) and personal data (age, sex, and training experience) were recorded before starting the swimming trials.
Swimming trials were performed in an indoor swimming pool (8 × 25 m, water temperature: 28 • C, water depth: 1.80 m). Swimmers maintained a depth of ∼0.8 m while performing UUS. The participants were instructed to use the push off from the wall only to obtain the correct water depth, and to generate the swimming speed by undulatory swimming only. All trials were filmed, and kinematics were obtained by two-dimensional motion analysis.

Standardization of kicking frequency
After independently warming up for 20 min including UUS, the participants performed three trials (15 m) of underwater dolphin kicks in maximum speed to determine their maximum kicking frequency. Between the trials they had a resting time of 5 min. The duration of three kick cycles were used to calculate the kicking frequency. The highest frequency of the three trials was set as individual maximum (100%).
The following trials were performed with submaximal effort (80% of individual maximum kicking frequency). The submaximal effort should provide an individual competitionlike intensity without risking to much muscle fatigue. The constant kicking frequency during all swimming trials should also ensure a consistent power output and prevent an impact of kicking frequency on UUS kinematics. Shimojo et al. (2014) showed no significant difference of kick efficiency (Strouhal number and Froude-efficiency) during swimming with submaximal kicking frequency (85%) compared to swimming with maximum kicking frequency. The calculated velocity was set by a waterproof metronome device (FINIS R Tempo Trainer Pro) which was clipped onto the swimming goggle. The synchronization of the kicking frequency to a metronome device has previously been shown to have no impact on kinematics, movement patterns and muscle activity of the lower extremities during UUS (Yamakawa et al., 2017). The swimmers performed three familiarization trials of 15 m with underwater dolphin kicks trying to synchronize their kicking frequency to the beat of the metronome device.

Swimming trials
The familiarization trials were followed by three trials of swimming with normal, restricted, and increased ankle joint flexibility, respectively. The participants were asked to swim as fast as possible while maintaining the set kick frequency of the metronome device.
The following conditions were tested and compared regarding different kinematic parameters: (1) Normal PF angle: participants swam with their natural ankle joint flexibility, (2) Restricted PF angle: plantar flexion was restricted by tape application on both feet by approximately 10% before swimming, (3) Increased PF angle: plantar flexion was increased by passive-dynamic stretching before swimming.
The condition "normal PF angle" was tested first on both test days. The order of the remaining conditions was individually randomized. Accordingly, there were two possible orders: On the second testing day the order of the last two conditions was reversed compared to the first testing day.
There were 3 min rest between trials and 10 min rest between sets. The application of the tape and the passive-dynamic stretching was performed during that resting time.

Taping
The amount of restriction was aimed to be high enough to produce measurable effects on swimming velocity and kick efficiency and low enough to prohibit unwanted effects on swimming technique. Considering previous studies which used either 30 or 4% (Willems et al., 2014;Shimojo et al., 2019), we aimed for a restriction in between of 10% of maximum plantar flexion.
Right before the swimming trials, the active PF angle was measured with a goniometer using the neutral zero method (Freiherr von Salis-Soglio, 2015). A waterproof elastic tape was used to restrict PF and all swimmers got taped by the same person (JK). The feet were held in a position of 80% of maximum PF angle while applying the tape as tightly as possible. The remaining restriction was supposed to result in 90% of maximum PF angle. After tape application, the active maximum PF angle was measured again to verify, that the sought PF restriction was achieved. If the tape loosened partially from the feet during swimming trials the tape application was renewed during the following resting time.

Stretching
Immediately before the swimming trials with increased PF a passive-dynamic stretching of the ankle joints was performed. Swimmers lay on the ground with straightened legs while the researcher (JK) moved the feet from maximum plantar flexion to maximum dorsiflexion within 5 s. This stretching was performed for 60 s and paused for 30 s. Before the first trial, the stretching was repeated three times. During the resting time between trials, the stretching was performed once to maintain the acute stretching effect. After every finished stretching session, the maximum active PF angle was measured. The plantar flexion angle increased on average by 6.87%. To counteract possibly reduced muscle activity after stretching, the swimmers performed three hops before each swimming trial.

Motion analysis
UUS trials of the participants were filmed with an underwater video camera (60 frames per second; GoPro HERO7, .

FIGURE
Experimental set-up (A) and motion analysis of swimming trial by tracking bony landmarks (B). P -P and P -P mark the highest and lowest points of the fifth toe during kicking cycles. P and P mark the start and end points of three kicking cycles.
GoPro Inc., San Mateo, USA) which was positioned 0.6 m below water surface and 10 m away from the starting point (perpendicular to the swimming direction). The camera was attached to a bar that was pressed against the wall of the pool to ensure a stable camera positioning while filming. The distance between camera and swimmers was 4 m. The recorded area of swimming was from 7.5 m to 12 m after push-off from the wall. A cone was placed 10 m from the starting point (push-off). Its width was used as reference for calibration of the swimming distance in the motion analysis program (see Figure 1). For motion analysis, six anatomical landmarks were marked with a waterproof pen on the lateral right side of the swimmer's bodies: trochanter major (hip), epicondylus lateralis femoris (knee), caput fibulae (knee), malleolus lateralis (ankle), calcaneus (heel) and caput ossis metatarsalis V (toe). The recorded videos were uploaded to a motion analysis program (Kinovea version 0.9.4) and landmarks of the swimmer's bodies were manually digitized (see Figure 1). A recent study reported that the Kinovea software is a valid and reliable tool that is able to measure accurately at distances up to 5 m from the object and at an angle range of 90-45 • (Puig-Divi et al., 2019).
The following kinematic variables were measured, respectively, calculated: (1) kicking frequency [Hz]: number of finished kicking cycles divided by duration of swimming,

Statistical analysis
Statistical analysis was performed using IBM SPSS (version 27). Shapiro-Wilk-tests were used for assessment of normal distribution. All kinematic variables were statistically normally distributed except minimum knee flexion angle during swimming with increased PF angle [W (10) = 0.83, p = 0.036, n = 10]. T-tests for dependent samples were used to compare the kinematics of the two separate testing days. An analysis of variance (ANOVA) for repeated measurements was applied to compare kinematics of the different swimming conditions for all variables, as it is robust against violations of the normal distribution. In case of a significant difference, a Bonferroni post-hoc analysis was conducted. The effect size f was evaluated as small (0.10-0.24), moderate (0.25-0.39) and large (>0.40) (Cohen, 1988). Pearson correlations r were used to determine the correlation between maximum PF angle and each kinematic parameter. Classification was made regarding the minimum levels of r: small (±0.1), moderate (±0.3) and large (±0.5). The level of significance was set at p < 0.05.

Results
There was no significant effect of testing day on UUS kinematics, thus the mean of both days was calculated and used for analysis of each tested condition (PF angle).
Maximum PF angles were significantly lower during swimming with restricted PF angle compared to swimming with normal or increased PF angle (Table 1). The effect size was evaluated as large (f = 4.36).
Kicking frequency, as set by the metronome, did not differ between test conditions as planned (Table 1).
Kick efficiency and horizontal swimming velocity were significantly smaller during swimming with restricted PF angle compared to swimming with normal and increased PF ( Figure 2). The effect size was evaluated as large (kick efficiency f = 1.54; swimming velocity f = 1.82). There were no significant differences regarding kick efficiency and horizontal swimming velocity between swimming with normal and increased PF. Regarding kick amplitude, the ANOVA for repeated measurements indicated a significant difference in kick amplitude between tested conditions [F (2,18) = 3.74, p = 0.044, η p 2 = 0.29]. Mean values for kick amplitude were highest during swimming with increased PF angle followed by swimming with normal PF angle and restricted PF angle (Table 1). However, Bonferroni corrected paired comparisons did not reveal significant differences between conditions. Minimum knee flexion angle was significantly smaller during swimming with restricted PF compared to swimming with increased PF ( Table 1). The effect size was evaluated as large (f = 0.79). There was no significant difference of minimum knee flexion angle between swimming with normal PF and restricted PF as well as between swimming with normal and increased PF.
Horizontal swimming velocity significantly correlated with body height in all tested conditions (restricted PF: r = 0.77, p = 0.010; normal PF: r = 0.66, p = 0.038; increased PF: r = 0.64, p = 0.046). Therefore, to account for interpersonal differences  in body morphology affecting swimming performance and to analyze the effect of maximum PF angle irrespective of body morphology, swimming velocity was normalized to body height for further analysis. Body height normalized swimming velocity significantly correlated with maximum PF angle (r = 0.538, p = 0.002) which is shown in Figure 3. There were no significant correlations between maximum PF angle and other kinematic variables.

Discussion
To our knowledge this is the first study that investigated the impact of both restricted as well as increased plantar flexion angles on UUS kinematics. In agreement with our hypothesis, swimming velocity and kick efficiency were affected by ankle joint flexibility. However, it was particularly the restriction of plantar flexion that was negatively affected, while the increase in plantar flexion did not further enhance swimming velocity or kick efficiency.
The negative effect of PF restriction on swimming velocity seems to be at least partially dose dependent. A study on the effect of ankle flexibility on dolphin kick performance in competitive swimmers, which restricted PF by tape application by 30% (Willems et al., 2014), has led to a 19.5% reduction in swimming velocity, while in our study PF restriction was smaller (10%) which has led to a smaller reduction in swimming velocity of 5.8%. In accordance, a small PF restriction of 4% in a study on ankle joint flexibility in UUS (Shimojo et al., 2019) resulted in a small reduction of swimming velocity of 4.7%.
Regarding the reason for the effect of PF restriction on swimming velocity, it has been suggested (Willems et al., 2014) that the area of the feet, which shifts the water backwards and generates the propulsive impulse, decreases with the restriction of the PF angle. Consequently, a higher knee flexion during the down kick has been observed which may be a compensatory strategy for restricted PF flexibility (Willems et al., 2014). The limited vortex generation due to restricted PF and the potentially greater frontal drag due to higher knee flexion were suggested to be the reason for the decreased swimming velocity. Previous studies also described a negative correlation of knee flexion and swimming velocity because of the higher frontal water resistance [r = −0.70 (Arellano et al., 2003); r = −0.53 (Wadrzyk et al., 2017)]. Our results point in the same direction as the reduction of swimming velocity with restricted PF angle was similarly accompanied by greater knee flexion when compared to swimming with increased PF angle (+1.2%). It is conceivable, that smaller reductions in PF lead to smaller or negligible changes in knee flexion. This is supported by the finding that a 6% reduction in PF (Wadrzyk et al., 2019) did not result in any significant changes in knee flexion angles.
It has also been suggested (Sugimoto et al., 2008;Cohen et al., 2012) that a limited inversion of the feet may cause a reduction in swimming velocity. In the present study, the measurement of the foot inversion angle was not possible due to the lateral and two-dimensional nature of the video analysis. Thus, we cannot test this assumption with our data. A previous study (Matsuda et al., 2021) detected no significant relationship between ankle inversion ROM and UUS velocity. However, this study analyzed the correlation between ankle inversion ROM and UUS velocity in fast and less fast swimmers and .
/fspor. . thus described interpersonal differences. Those interpersonal differences in UUS velocity can be affected by a huge variety of variables not allowing to conclude on the effect of ankle inversion ROM on UUS velocity within one person. While the combination of maximum plantar flexion and inversion may be important for a perfect propulsion during UUS, we are confident that there is a causal relationship between PF restriction and decreased swimming velocity in our study. In agreement with the literature, the restricted maximum PF angle could have caused a greater frontal drag (Ungerechts et al., 2009). Additionally, the smaller range of motion could have resulted in a limited flipping action of the feet during the down kick so both the size and the rotation velocity of the generated vortices could have been reduced, which, in turn, would have decreased the swimming velocity and kick efficiency. Also, the tape material could have changed the streaming characteristics of the water as well as the vortex generation which could have contributed to the decreased swimming velocity and kick efficiency. However, the extent of this possible impact cannot be determined yet.
In contrast to the negative effect of PF restriction on swimming velocity, it seems that an increase in PF by stretching intervention does not further enhance swimming velocity. It is conceivable, that the increase in PF angle compared to the normal condition may have been too small to result in significant changes in swimming velocity and kick efficiency. It is also possible that other three-dimensional movements of the lower limbs are crucial to achieve higher UUS velocity, as it has been shown that e. g., the peak angular velocities of hip internal and external rotation were significantly correlated with UUS velocity (Matsuda et al., 2021). However, body-height normalized swimming velocity highly significantly correlated with maximum PF angle in the present study which indicates that the ankle joint flexibility affects swimming velocity. This result contrasts with the findings of Willems et al. (2014) who did not find a significant correlation between swimming velocity and PF angle. They assumed that the ankle joint flexibility is a neglectable factor regarding swimming velocity compared to other determinants like muscle power or water drag. While other determinants may affect swimming velocity to a greater extend, we suppose that PF angle has at least a small impact on swimming velocity, which may decline with increasing ankle flexibility. Thus, there can be an optimal condition of plantar flexion flexibility beyond which no further gain in swimming velocity and kick efficiency is realized. Furthermore, the normal PF angles of our participants were with 50 • highly above average. It is possible that in swimmers with lower initial values of PF angle a larger effect on swimming velocity and kick efficiency may have been measurable which needs to be confirmed in future studies. However, especially in longer races even small improvements of kick efficiency can have a particular impact on overall performance as a higher kick efficiency can result in lower energy cost and therefore faster racing times (Zamparo et al., 2020). Over a 400 m short course race a time improvement of ∼2 s can be calculated based on the within our study detected improvements in kick efficiency and swimming velocity with increased PF (compared to normal PF), when swimming with an intensity of 80% and assuming an UUS distance of 10 m excluding start and turn push offs.
Considering the presented results, it is conceivable that particularly athletes with low or average maximum plantar flexion angles could benefit from of a long-term ankle joint flexibility program to improve their UUS and overall performance.
Although the study was conceived and performed with care to obtain objective, valid and reliable data, there are some points to discuss that may limit the interpretation of the results. The present study investigated acute effects of ankle joint flexibility changes only. However, long-term increases in plantar flexion flexibility may differently affect UUS velocity. For instance, we observed a higher knee flexion, possibly resulting from an acute compensatory mechanism in response to restricted PF but long-term impacts of limited PF on knee flexion cannot be derived from the presented results and may differ from acute effects. Moreover, the elastic material of the tape may not have fully restricted the PF angle during swimming because of the high passive forces underwater. A non-elastic tape could have reduced this potential discrepancy between maximum PF angle on land and during swimming. However, when applying a nonelastic tape the participants of Shimojo et al. (2019) reported pain during swimming. Furthermore, pilot-trials of the present study demonstrated that a non-elastic tape was not waterproof, so the tape got loosened during the swimming trials and PF angle was no longer restricted. For this reason, a waterproof elastic tape was used in the present study and was sticked as tightly as possible onto the skin. In addition, exact measurement of PF angle and inversion during swimming was not feasible due to two-dimensional video analysis and combined movement of PF and inversion during down kick. Thus, future studies should consider a 3D movement analysis to capture foot inversion in addition to PF. Besides, an underwater camera was used to film the swimming trials. While a linear field of view was set, there was a slight distortion at the outer frame of the video. To counteract this effect, analyzed swimming trials were always in the center of the video. Lastly, the study size may have been too small to find small yet significant changes of e. g., kick amplitude and a resulting impact on UUS performance as well as gender-specific effects.
Further research is necessary to determine the magnitude of the impact of ankle joint flexibility on swimming velocity and kick efficiency as well as the threshold level of PF angle upon which swimming performance does not further improve. Kinematics between swimming with normal and increased PF angles should be tested and compared particularly in swimmers with impaired ankle flexibility to observe the effects of increased PF angle on UUS performance.

Implications for practice
Since success in competitive swimming races is often determined by milliseconds, factors affecting swimming stroke efficiency must be identified and optimized to improve the athlete's performances even by marginal gains. As reduced ankle joint flexibility impairs UUS velocity, we recommend that particularly swimmers with low or average PF angles should consider implementing ankle joint flexibility exercises in their training regime to improve their UUS performance.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Ethics statement
The studies involving human participants were reviewed and approved by the Ethics Committee of the Faculty of Humanities and Social Sciences of the Humboldt-Universität zu Berlin. The patients/participants provided their written informed consent to participate in this study.


s2fieldsofstudy: Biology Medicine

text:
Cannabidiol Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in the Inflammatory Microenvironment via the CB2-dependent p38 MAPK Signaling Pathway

Background and Objectives Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment. Methods and Results BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and presented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs’ vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively. Conclusions CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.

Introduction
Long-term inflammatory states suppress bone reconstruction (1), controlling inflammation is of foremost importance to prevent inflammation-induced bone loss (2). During physiological bone healing, bone marrow mesenchymal stem cells (BMSCs) develop and differentiate into mature functional bone cells under the activation of cytokines (3) to maintain bone tissue homeostasis and repair (4). However, differentiation potential and functional status of stem cells were suppressed in exaggerated inflammatory microenvironment (5,6). Therefore, agents that are characterized by anti-inflammatory property may promote osteogenic differentiation of BMSCs in the inflammatory microenvironment.
Cannabinoids have been used for thousands of years due to their multiple pharmacological functions, such as analgesic and anti-inflammatory effects. But psychoactivity makes them controversial to use (7). The isomers Δ-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) are two main cannabinoids (8). Unlike THC, non-psychoactive property of CBD avoids its abuse and public health problems. CBD possesses wide range of pharmacological functions including anti-epilepsy, anti-anxiety, anti-tumor, and of note anti-inflammation effects (9), such as gastrointestinal inflammation (10), non-alcoholic steatohepatitis (11), dermal inflammation (12) and osteoarthritis (13). Besides, some clinical studies have also shown that CBD promoted healing of non-infectious arthritis (14) and took beneficial effect on bone metabolism (15). Based on the anti-inflammatory effects of CBD, we hypothesized that CBD may present osteogenic potential in inflammatory conditions. Lipopolysaccharide (LPS) is the main toxic factor generated by bacteria during infectious bone diseases (16). So, we applied LPS to induce inflammatory microenvironment, and investigated the osteogenic differentiation capacity of LPS-impaired BMSCs and related mechanisms by CBD treatment.

Cells isolation and culture
BMSCs were isolated from five-week-old male C57BL/6 mice (Animal Experimental Center of Chinese People's Liberation Army General Hospital, Beijing). First, we separated the femur and washed the bone marrow cavity repeatedly to collect the cells. Then, cells were inoculated in α-minimum essential medium (α-MEM; Gibco, USA) containing 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin/streptomycin in a humidified incubator at 37℃ with 5% CO2. An optical microscope (Olympus, Japan) was used to daily observe the morphology and growth of the cells. Finally, the third to fifth passage cells (P3-5) were used for subsequent experiments.

Stem cells identification
P3 BMSCs were washed for three times, digested and collected for the following experiments. First, representative surface markers were analyzed by flow cytometry. After labeled with antibodies of CD11b, CD29, CD31, CD34, CD44, CD45, CD73, CD90, and CD105 (BD Biosciences, USA), the BMSCs were incubated at 4℃ for 30 min. After which, cells were washed with PBS, checked by flow cytometry (BD Celesta, USA) and finally analyzed using CytExpert 2.0 software.
Quantitative real-time polymerase chain reaction (qRT-PCR) P4 or P5 BMSCs were seeded in 6-well plates and cul-   (17). The 2 -Δ ΔCt method was adopted to analyze the relative gene expression and primers sequences are presented in Table 1.

Statistical analysis
Data are presented as means±standard deviations (SD) and were analyzed by GraphPad Prism Software (version 9.0, USA) and SPSS (version 23.0, USA). The Shapiro-Wilk test was used to evaluate the normality of data. Homogeneity of variance was tested using Levene's test. One-way analysis of variance (ANOVA) test was used for multiple sample comparison. p＜0.05 (two-sided) was considered statistically significant.
Next, we detected the effects of CBD on BMSCs' viability. CCK-8 assay showed no statistical differences in absorbance values among different doses of CBD, after 72 hrs of administration ( Fig. 2A), which indicated that CBD took no obviously toxic effects on BMSCs vitality in the tested concentrations.

CBD downregulates LPS-induced inflammatory response
Previous study has demonstrated that CBD inhibits the inflammasome in human gingival mesenchymal stem cells (19). Here, LPS (10 μg/ml) (20) was applied on BMSCs to establish the inflammatory microenvironment, and we investigated whether CBD could downregulate LPS-induced inflammatory response of BMSCs. qRT-PCR results showed that mRNA expression levels of tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) were significantly increased in LPS-treated BMSCs. As expected, CBD pretreatment reversed this increase of aforementioned inflammatory cytokine expression levels (Fig.  2B). Also, the efficacy of 2.5 and 5 μM of CBD were more obvious than 0.5 μM CBD to decrease TNF-α and IL-6 mRNA expression levels (p＜0.05), and no statistical difference was observed between 2.5 and 5 μM CBD groups.
Overall, CBD could effectively decrease LPS-induced inflammatory response.
CBD promotes osteogenic differentiation of BMSCs in the inflammatory microenvironment Subsequently, we wondered whether CBD could promote osteogenic differentiation of injured BMSCs in LPS-induced inflammatory environment. ALP is considered an important indicator in early osteogenesis (21). Thus, we detected the ALP activity of BMSCs after osteogenic induction and found that CBD significantly increased ALP activity in a dose-dependent and saturated manner. And 2.5 and 5 μM CBD groups were comparable at 3, 5 and 7 days (Fig. 3A), so 2.5 μM of CBD were selected as the intervention dose in the following experiments.
Second, we determined osteogenesis-related protein expression levels in BMSCs using western blot. The expression levels of Runx2, an essential transcription factor in early osteogenesis (21), were declined in the LPS group, while CBD intervention reversed this decrease. Meanwhile, the protein expression levels of ALP were consistent with the result of Runx2 protein. In addition, CBD pretreatment could also recover the OCN expression levels, a crucial marker expressed in late stage of bone formation (21) (Fig. 3B and 3C). The original blots are shown in Supplementary Fig. S1.
Last but not least, we detected the mRNA expression levels of Runx2, ALP and OCN, the results were consistent with the results of western blot (Fig. 3D). Collectively, these findings demonstrated that CBD reversed the attenuated osteogenic differentiation of BMSCs in the LPS-induced inflammatory microenvironment.

CBD activates the CB2-dependent p38 MAPK pathway
To explore the molecular mechanism of CBD administration on BMSCs osteogenic differentiation, we first detected whether CB1 and/or CB2, essential cannabinoid receptors expressed in osteocytes and related to bone metabolism (22,23), participated in osteogenesis process of CBD administration. We found that both CB1 and CB2 restrained when co-treated with LPS (Fig. 4A). While, CBD significantly promoted CB2 expression in the inflammatory microenvironment, indicating that CBD might initiate intracellular effects via the activation of CB2. The original blots are shown in Supplementary Fig. S2.
Mitogen-activated protein kinase (MAPK) family is a downstream signaling pathway of cannabinoid receptor, Fig. 4. CBD activates the CB2-dependent p38 MAPK pathway. (A) BMSCs were pretreated with LPS (10 μg/ml) for 12 hrs, then CBD (2.5 μM) were added and cultured for another 12 hrs. Western blots and quantitative analysis were performed for determining CB1 and CB2. β-actin was used as the internal control (n=3). (B) BMSCs were pretreated with LPS for 12 hrs, then CBD and AM630 (10 μM) were added as indicated and incubated for another 12 hrs. Western blots and quantitative analysis for p-p38 and p38 were performed. β-actin was used as the internal control (n=3). CB1: cannabinoid receptor 1, CB2: cannabinoid receptor 2, p-p38: phosphorylated p38. Data are presented as means±SD. *p＜0.05 compared with the LPS group; # p＜0.05 compared with the LPS plus CBD group. and p38 is an important MAPK isoform associated with inflammatory response (23). We found that p-p38 decreased in LPS-induced BMSCs, but CBD administration increased its expression (Fig. 4B). Furthermore, when inhibited CB2 with AM630, a selective antagonist of CB2, the effects of CBD activated p38 MAPK signaling pathway were blunted. The original blots are shown in Supplementary Fig. S3. Together, these results suggested CBD activates the CB2-dependent p38 MAPK pathway.

CB2-dependent p38 MAPK signaling pathway is required for CBD promoting BMSCs osteogenic differentiation
Next, to confirm the effects of the CB2-p38 MAPK signaling pathway on CBD promoting osteogenic differentiation of BMSCs, we used AM630 co-treated with CBD. Then we found that both the protein and mRNA levels of Runx2, ALP and OCN decreased compared with CBD group, indicating that CBD promoted osteogenic differentiation of BMSCs, at least partially, via CB2.
Additionally, SB203580, a selective p38 inhibitor, decreased both protein and mRNA levels of Runx2, ALP and OCN compared with CBD group, indicating that CBD en-hanced osteogenic differentiation of BMSCs, at least partly, via p38 MAPK signaling pathway. Interestingly, we also found that the decreases of ALP and Runx2 protein and mRNA expression levels in the AM630 group were more obvious than the SB203580 group (Fig. 5A∼C). The original blots are shown in Supplementary Fig. S4. Moreover, alizarin red staining (Fig. 5D) and semi-quantitative analysis (Fig. 5E) showed that the application of CBD increased the deposits of calcium nodules compared with LPS-injured BMSCs, while co-treated with AM630 or SB203580 reduced the formation of calcium nodules, demonstrating that at the final stages of differentiation, CBD promotes osteogenic differentiation of BMSCs in the inflammatory microenvironment via the CB2-dependent p38 MAPK signaling pathway.

Discussion
The result of our study, for the first time demonstrated that CBD could promote osteogenic differentiation of BMSCs in LPS-induced inflammatory microenvironments. The major findings are: (1) CBD intervention down-regulated TNF-α and IL-6 expression levels and Fig. 5. CB2-dependent p38 MAPK signaling pathway is required for CBD promoting osteogenic differentiation of BMSCs. BMSCs were pretreated with LPS (10 μg/ml) for 12 hrs. Then CBD (2.5 μM), AM630 (10 μM), and SB203580 (20 μM) were added as indicated and co-cultured with LPS for 7 days. (A) Western blots were performed for detecting the protein expression levels of Runx2, ALP, and OCN. β-actin was used as the internal control (n=3). (B) Quantitative analysis of (A). (C) BMSCs were harvested for measuring the mRNA expression levels of Runx2, ALP, and OCN using qRT-PCR. β-actin was used as the internal control (n=5). BMSCs were co-treated with these agents for 14 days and then alizarin red staining was performed. Representative images (D) and semiquantification (E) of alizarin red staining after incubation for 14 days (Scale bar, 100 μm). Data are presented as means±SD. *p＜0.05 compared with the CBD plus LPS group. # p＜0.05 compared with the CBD plus AM630 group.
Appropriate pro-inflammatory signaling is necessary in the early stage of fracture healing. However, chronic inflammation hazards to the osteogenic environment (24). Thus, inhibiting inflammation is essential to improve the osteogenic differentiation environment for BMSCs. Recent studies found that CBD, which is already approved by the FDA for refractory epilepsy treatment in children (25), could promote stem cells odontogenic and osteogenic differentiation (26,27). Additionally, scaffolds combined CBD enhanced mesenchymal stem cell recruitment and regeneration of critical-sized bone defect (28) in non-inflammatory environment. Our study further demonstrated that CBD promoted BMSCs osteogenic differentiation in LPS-induced inflammatory environment based on the following results: 1) CBD intervention enhanced ALP activ- ity; 2) up-regulated osteogenesis-related gene expression levels of BMSCs; 3) increased the expression levels of osteogenesis-related proteins.
It has been established that endogenous cannabinoid system is a key modulator of bone mass (29,30). For example, CB1 or CB2 knockout mice developed osteoporosis and low bone mass phenotype (31). It was reported that inflammatory environments reduced cannabinoid receptors expression in stem cells (32), which might be associated with impaired cell function. Our results showed that LPS administration decreased both CB1 and CB2 expression, while CBD intervention reversed the down-regulation CB2, but took no effects on CB1 expression, thus excluding the possibility of CBD promoting osteogenic differentiation in inflammatory environment via CB1. However, research has implied that CB1 enhanced the osteo/dentinogenic differentiation ability of periodontal ligament cells (32). One possible explanation for this discrepancy is that we used the different type of stem cells. In fact, CB1 mainly expresses in central nervous systems, while CB2, mainly expressed in peripheral tissues, involves in regulating bone volume (15). Also, CB2 receptors are more densely distributed in osteocytes than CB1 (23,29). Furthermore, CB2 has a higher affinity than CB1 to CBD and plays crucial effects in CBD promoted biomineralization (33). In fact, CB2 activation increases the expression levels of Runx2, ALP and bone sialoprotein (BSP) of osteocytes in non-inflammatory microenvironments (21). In our study, when applied AM630, a specific CB2 inhibitor, the effects of CBD on promoting osteogenic differentiation of BMSCs in LPS-induced in-flammatory microenvironments were blunted. Collectively, these data indicated CBD promoted BMSCs osteogenic differentiation in inflammatory microenvironments via CB2.
The MAPK family includes three isoforms: extracellular signaling regulatory kinase (ERK), c-jun amino-terminal kinase (JNK), and p38 kinase (p38 MAPK), which are involved in inflammatory processes (32). In our findings, LPS inhibited the activation of p38 MAPK signaling pathway, while CBD activated p38 MAPK signaling pathway in an inflammatory environment, which was consistent with previous studies showed that CBD promoted MG-63 cells osteogenesis differentiation through p38 MAPK pathways in non-inflammatory microenvironment (34). Cannabinoids and their correspondent receptors could activate downstream MAPK isoforms (35). It was reported that overexpression of CB2 in osteoporosis increases p38 phosphorylation and mineralized extracellular matrix deposition (36). In contrast, when blocking CB2 with AM630 or siRNA targeted CB2, MAPK signaling pathway was suppressed (26,31). We also found that p38 MAPK was restrained after CB2 inhibition using AM630, indicating that downstream p38 MAPK is effect-dependent on CB2. Further, p38 MAPK directly regulates downstream osteogenesis-related important target genes, such as Runx2 and osterix (37). Meanwhile, enhanced ALP activity and osteogenesis-related target genes expression of MSCs were also associated with MAPK activation in non-inflammatory environment (26,27). We found that CBD intervention activated p38 and increased osteogenesis-related mRNA and protein expression levels in inflammatory environment.
Moreover, when co-treated with p38 MAPK selective inhibitor, SB203580, the effects of CBD-promoted notably osteogenic differentiation of BMSCs diminished. Taking together, CBD promoted osteogenic differentiation of BMSCs in inflammatory microenvironment via the CB2-dependent p38 MAPK pathway.
This study also has some limitations. First, although we concluded that CB2 was the primary receptor in CBD-promoted osteogenesis, however, CB2 specific inhibitors did not completely inhibit CBD osteogenesis effects, indicating that CBD may also promote osteogenesis through other mechanisms. G protein-coupled receptor 55 (GPR55) is another important cannabinoid receptor, also participates in CBD-mediated bone mass regulation (38). So, other mechanisms, such as GPR55 signaling pathway, could not be elucidated. Second, the concrete experimental data linking osteogenesis differentiation of BMSCs with CB2 dependent p38 MAPK signaling activation remain unclear. Finally, we did not perform in vivo experiments. Further in vivo researches should be performed to clarify the osteogenesis potential of CBD in inflammatory microenvironments.
Overall, we verified the efficacy of CBD to promote osteogenic differentiation of BMSCs in the LPS-induced inflammatory microenvironment via the CB2-dependent p38 MAPK pathway. As a natural active drug with anti-inflammation property, CBD could be considered as a potential strategy for treating inflammatory bone diseases.